CA2028146A1

CA2028146A1 - Composition containing labeled streptococcal antibody, test kit and assay using same

Info

Publication number: CA2028146A1
Application number: CA002028146A
Authority: CA
Inventors: Chester H. Warren Iii; Gary L. Snodgrass
Original assignee: Eastman Kodak Co
Current assignee: Eastman Kodak Co
Priority date: 1989-03-01
Filing date: 1990-02-13
Publication date: 1990-09-02
Also published as: FI905350A7; EP0413810A1; FI905350A0; JPH03501653A; WO1990010232A1; KR920700402A

Abstract

An aqueous immunological composition comprises a detectably labeled antibody to a Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant. This composition provides improved background and spot quality in assays for Streptococcal antigens. Such advantages are particularly evident with assays carried out using filter membranes to capture the immunological product. The composition can be included in a diagnostic test kit for use in the assay.

Description

f`~ V h- U .~

2 PCT/US90tO0761 COMPOSITION CONTAINING LABELED STREPTOCOC~AL
ANTIBODY, TEST gIT AND ASSAY USING SAME
Fiel~_Q~_~he Invention This invention i8 related to an immunological composition containing a labeled antibody to a Streptococcus or~anism. It also rela~es to an immunological diagnostic test kit including the composition. The invention i8 useful in diagnostic methods for determining Streptococcal organisms.
~ackground of the Inven~ion Organisms classified as gram positive bacteria, such as group specific Streptococcus, are ~nown to be pathogenic in humans. Group A organisms are primarily responsible for causing B-hemolytic pneumonia, scarlet fever, rheumatic ~ever, cardiac sequelae, glomerulonephritis, septic ~ore throat and puerpueral sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagno6e its presence at an early ~tage ~o that an appropriate course of treatment can be pursued. In other words, it is highly desirable to have a highly sensitive assay ~o that infectious agents can be detected at low concentration.
The antigen-antibody reaction i8 the basis for all immunological test methods. Certain proteins known as antibodies are produced by mammals and humans in re~ponse to the presence of an antige~, or ~oreign substanc.e, which can be another protein or a carbohydrate. This normal body response to a ~oreign substance has led to the development of a ~umber of diagno~ ic technique6 to detect drugs, diseases or other physiological conditions, including the pre~ence of Streptococcal organi~ms.

.L
WO90/10232 PCTtUS90/00761 , For example, in an in ~i~EQ as~ay for Streptococcal organi~ms in a test specimen, the presence of the antigen from the organi~m i8 detected by contacting the specimen with it6 immunological counterpart, that is an antibody. If the antigen iB
present, the resulting immunological comple2 wlll Porm a~d can be detected using appropriate techniques and reagent~.
Such complexes are ~etected in a number of ways. Generally, one or more participants in the reaction is detectably labeled. That i8, it is either chosen because it is inherently detectable, or a detectable moiety (for example, an enzyme, radioisotope, chromogen or fluorogen) i~ incorporated therein in some manner. Many assays (for example, that known as ELISA) today utilize enzyme~ as detectable moieties because of the con~enience they provide in requiring minimum equipment and ~kills needed for the a88ay3, as well as improved sensitivity i~ some cases.
One significant problem encountered in ~any analytical procedures which utilize immunological reactions is the occurrence of non-~pecific binding reactions. For example, immunological reactantE, ~uch as antibodies or antigens, may indiscriminately react with other proteins, carbohytrates or chemical or biological materials for which they are not ~pecific. They ~ay als~ ~eact with each other and clump together thereby inhibiting speci~ic binding with a molecule ~or which they have specific reactivity. In addition, i~ the assay~ are carried out using solid support~ o~ some type (such as ~embrane8, gla88 tubes, plates, beads or fibers), the speeific binding specieæ may also non-specifically bind ~o such mater;als because of the nature of h ~ ~
-woso/lo~32 PCT/US90/00761 chemical groups on the ~urfaces. This can be particularly troubling if the immunological complex i8 detected on a solid support in a cestain region only, because unwanted reactions are ob~ervable as background signal. If this background i~ too high, the signal due to the de~ired immunological reaction is obscured or misread. Low antigen concentrations are undetectable, and a~ay 8ensitiYity is lowered.
Polyamide ~embranes, in partit:ular, are susceptible to nonspecific interactions with antibodies. It is known that non-specific bindin~ to solid surfaces (such as polyamide~) can be mini~ized by coating them with proteins, such as ca~ein or bovine serum albumin. I~ copending US-A-4,828,9B0, it is disclosed that non-~pecific interactions in agglutination assays can be minimized by coatin~-microporous membranes used in thoQe assays with certain low pI proteins or carbohydrates. Moreover, copending U.S.S.N. 206,257 (filed June 13, 1988 by Warren III et al) describes the use of water-soluble proteins or carbohydrates with labeled antibodies in vrder to lower background and improve as~ay 8en8iti~ity .
While the no~ed inventions represent important advances in the art, it has been observed that it would be Yery helpful if background was reduced further in a88ay8 for Streptococcal - organisms. In particular, in instances where the a88ay i8 carried out in a distinct region of a microporous fi~tration mem~rane, the area around that region sometimes exhibits clumps of labeled antibody which ob~cures the as~ay signal, can lead to ~alse positive results and can hinder assay determinations in the distinct region.

2 ~
woso/1o232 PCT/US90/00761 Summarv of the InventiQn The problems mentioned above have been overcome with the use of an aqueou~ i~munological composition comprising a detectably-labeled antibody to a Streptococcal antigen, and a water--~oluble or water-dispersible nonionic sur~actant.
Moreover, an immunological diag~nv~tic test kit u~eful for the determination of a St:reptococcal organi~m comprises: .
a. one or more reagent~ for e~tracting Streptococcal antigen from a Streptococcal organism, and b. the aqueous immunological composition described above.
This invention also provides a method for the detection of a Streptococcal organism comprising the steps of:
A. contacting a specimen suspected of containing an extracted Streptococcal antigen with the aqueous immunological composition aescribed above, to form a detectably-labeled immunological complex, i -B. detecting the presence of the complex as anindication of the pre 8 ence of the anti gen in the specimen.
The pre~ent invention provides a compo~ition which enable~ one to have a highly ~ensitive diagnostic a~say for Streptococcal organi~ms using a detectably labeled antibody. The improved 30 ~ensitivity and ~pecificity of . the assay are acco~plished by using the labeled antibody in admi~ture with one or ~ore water-~oluble or water~dispersible no~ionic ~ur~acta~ts~ It i~ not under~tood how these surfactants provide the 3~ improvement, but it i~ clear that background i~

~;~090/10232 PCTtUS90/00761 lowered and the clumps of labeled antibody formed in back~round re~ions of a sub6trate are noticeably reduced. Thus, spot quality i8 improved when the test i8 carried out in a "8pot" or region of the substrate. Moreover, a~ay sensitivity i8 not adver~ely affected, and may be impro~ed in ~any instances because lower concentrations of antigen are readily detected.
Detailed De~c~i~tion o~ ~he Inven~ion The present inventlon can be u~ed ~o rapidly detect the preæence of a Streptococcal organism in a biological ~pecimen ~rom a human or animal host.
Generally, such organisms are detected by determination of the presence of extracted Streptococcal antigens, such as the carbohydrate antigens extracted from Streptococcal A, B, C or G
group organisms. Streptococcal A antigen is most preferably detectable with this invention.
Biological samples which can be BO assayed ~o include any solution, specimen or sample which is ~uspected of containing Streptococcal organi~ms or e~tracted Str.eptococcal antigens. Generally, such organisms are found in human mucous and ti~ue:
culture~ f rom the mouth, throat or na~al pa~saEes.
The specimens can be collected using ~uitable procedures. For example, a throat specimen is generally collected on a swab, and can be te~ted with or without pretreatment (for example, filtration~ to remove unwanted debris or interferents.
While some analytical procedures may be ~uitably carried out using whole Streptococcal orga~ism since ~ome antigenic ~ites may be external to the bacte~ial cells, and thus, ava lable for immunological reaction, it i~ preferred that the assay be carried out with egtsacted antigen.

woso/lo232 PCT/US90/00761 ,~-Streptococcal antigen extraction can be accomplished using a number of known procedure~. For e~ample, extraction can be carried out using hot formamide, autoclaving in the pre8ence of ~Cl, variou~ enzymes (see ~S-A-4,6l8,576, or by generation of nitrou~ acid according to US-A-4,673,639 U8i~g one or more extracting reagent~. A preferred extraction technique is described in copending US-A-4,808,524.
In that preferred extraction technigue, a specimen suspected of containing Streptococcal orgasisms (such as from a patient) i8 subjected to the preferred extraction composition. For example, a swab containing a throat culture can be put into a container containing the extraction composition and l~ incubated therein for a suitable period of time. If desired, centrifugation can be carried out to remove extraneous matter or the resulting extract can be neutralized.
Extracted Streptococcal antigen i8 contacted with the aqueous immunological composition of this invention to form a detectably-labeled immunological complex. This composition comprises a detectably labeled antibody to the antigen. By detectably labeled i8 meant that the antibody can be directly detected u~ing appropriate detection equipment and procedure~, or it can be detected after further reactio~ of the label with one or more appropriate reagents. The label can be an inherent portion of the antibody molecule, or a separate moiety ~ttached thereto. Use~ul labels include radioisotope~, chemiluminescent moietie~, chromogen~ or fluorogens, phosphoresce~t moieties, enzymes, enzyme cofactors, and other6 known to one s~illed i~ the art.
Generally, the labeled a~tibody i8 water-soluble or 35 water - di~perBible, 80 the label ~u~t not insolubilize the antibody.

Another class of label~ are what are termed in the art as ~pecific binding material~ which will specifically bind with a corresponding receptor material. The resulting complege3 are comparable to immunological comple~es. E~amples of specific binding comple~es include avidin-biotin and sugar-lectin. Thus, the Streptococcal antibody can ~e labeled with avidin, biotin, a sugar, a lectin, or even another antibody or antigenic material not 8pecific ~or Streptococcal anti~ens. Detection of the i~munological complex i~ accomplished by reacting it ~ith the receptor corresponding to the label, which receptor ha~ a detectable moiety (for example, a radioisotope, enzyme or cofactor).
Radioisotopes and enzymes are preferred labels with enzymes being most preferred.
Representative examples include peroxidase, alkaline pho~phatase, urea~e, glucose oxidase, ~eta-glucosidase and others known in the art.
Peroxidase and alkaline phosphatase are preferred with peroxidase being most preferred.
Labeled antibodies can be prepared using ~nown procedures. ~any are commercially available.
Others are not co~mercially available, but they are prepared using known procedures (~ee, for example, ~thods in ~nzvmolo~Y, 92, Immunochemical Techniques, p.777, Langone and Van Vunakis, Eds., 1983 for preparing radiolabeled antibodies, EP-A-0 201 079 and ~S-A-4,276,206 for preparing biotinylated a~tibodiee). An enzyme-labeled antibody i~ generally prepared by derivatizing the enzyme, purifying the enzyme derivative, reacting the terivati~e with the antibody, and purification and characterization of tbe re~ulting conjugate. A number o~ procedure~ are described in the following reference~: ~oshitake, WO90/10232 pcT/usso/oo76 ~r. l. ~iochem.. 101, 395 (1979), Nakane et al, ~.
~is~ochemistrY and ~Ytochemi~trY. 22, 1084 (1974) and Avramea~, ~lL_~c. ~kim Biol., 50, 1169 (1968).
The amount of labeled antibody present i~
the composition of this invention will vary depending upon the target ligand, the dye-forming co~position ~sed ~here an enzyme label is u~ed), the particular label used ant other ~actor~ of a particular assay.
Generally, however, it i~ present in an amount of at least about 0-1 ~g/ml, and preferably from about 1 to about 20 ~g/ml.
The labeled antibody and unlabeled antibody (described below) used in the practice of this invention can be individually or collectively monoclonal or polyclonal. They ca~ be obtained from several commercial sources (such as Biochemed, Immunosearch or Ventrex), or prepared using ~tandard technigues.
The second critical component of the immunological composition is one or more water-soluble or water-dispersible nonionic BurfaCtantæ. Such ~urfactants are generally soluble or dispersible in water in an amount of at lea~t about 1 mg/ml.. U~eful sur~actants include, but ase not limited to, polyoxyethylene ethers such a~ thoae sold as TritonTM nonionic surfactants (for example, TritonTM X-100 and TritonTM N101), or a~
NonidetTM or BrijTM nonionic ~urfactants (such as NonitetTM p_40 and BrijTM 35), polyoxyethylene-30 sorbitan deri~atives ~uch as those sold as TweenTMnonionic surfactants (for example TweenTM 20 or TweenTM 40), ant polyglycol ethers such as those ~old as TergikolTM nonionic surfactants (for example TergitolTM NPX and NP-7). Other use~ul material~ would be seadily apparent to one ~illed in V i WO 90/1 0232 PCI /US90/01)7~1 _9 _ the art, especial~y after consulting the standard reference for surfactants, M~Cut~heon's ~mul~ifiers and Deter~e~ts, 1986 Edition, McCutcheon Division, Publi~hing Co., &len Rock, N.J., as wel:l a~ the Sigma Chemical Company catalog entitled "Biochemicals Organic Compounds for Research and Diagno~tic Reagents" for 1989, pp. 333-335, Particularly useful ~urfactants are the polyo~yethylenesorbitan derivatives such as the monolaurate, ~onooleate, monopalmitate, mono~tearate and other derivatives. The monolaurate derivative is most preferred. This material can be purchased as TweenTM 20 nonionic surfactant.
The nonionic surfactants are present in the immunological composition of this invention in an amount of at least about 0.05, and preferably from about 0.1 to about 0.5, weight percent (based on total composition weight).
The immunological composition optionally, but preferably, also contains a number of other component~ which improve assay performance. For e~ample, it can include a water- oluble protein or carbohydrate which has a low pI. The term pI (or isoelectric point) is known as the ph at which thexe is an equal ~umber of positive and negati~e charges in a molecule ~o that the molecule i6 neutral in charge. The pI can be measured using standard materials and procedures. Such materials include, but are not limited to, casein deri~atives or other protein derivatives which ase negatively charged (~or example 3uccinylated ca~ein, glutarylated easei~, succinylated bovine ~erum albumin and ~uccinylated coll~gen) and cellulosic derivative~ (such aæ
carbogymethy1 cellulo~e and car~oxyethyl cellulose).
Succinylated casein is preferred.

Wogo/10232 pcT/usso/oo761 ~-0 The composition can al80 include one or more phenol~ which impro~e its keeping stability. Useful phenols are described, for example, in US-A-4,828,983, and include ~ biphenol, 4'-hydro~yacetanilide, ~-methoxyphenol, chlorphenol red, ~-cre~ol, ~-methoxyphenol, vanillin~
4-chloro-3,5-dimethylaminophenol and ~-hydroxybenzoic acid. The phenol~ ~-methoxyphenol and 4'-hydroxyacetanilide are preferred, with 4'-hydro~yaceta~ilide being mo~t preferred. The amount of phenol pre~ent in the composition i6 generally at least about 0.001 weight percent.
One or more buffers are generally included in the immunological composition. Any buffer, in an appropriate amount, which will effectively maintain composition pH in the range o~ from about 7 to about 9 are useful. Representative buffers include, but are not limited to, 3-(N-morpholino)propanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-2-ethane-sul~onic acid and N-tris(hydroxymethyl~methyl-2-aminoethanesulfonic acid.
. The complex formed between Streptococcal antigen and detectably labeled antibody can be detected using suitable procedures and equipment depending upon the type of label used.
Where the label is an enzyme, the eomplex is contacted with ~uitable substrates and one or more dye-~orming reagents. Xn 80me insta~ces, the substrate itself provides a dye. Such sub~trates include benzidine or deri~atives ~heseof, 2,2'-azi~o-di-(3-ethyl-benzthiazolone-6-sulfonic acid), phenol red, Q-phenylenediamine, pyrogallol, 4-aminoantipyrine, bromopyrogallol red and other~
~nown in th~ art. Alternatively, a hydrogen donor 35 a~d an elec~ron acceptor can be combined to provide a detectable species (for example, as shown in US-A-4,260,679).

~ 90/10~32 PCT/US90/00761 Preferably. the label i~ peroxida~e ~nd the dye-providing reagents include a leuco dye which provides a dye in the preæence of peroxidase and hydro~en peroxide. Useful leuco dyes include imidazoles, arylmethanes and other~ kno~n in the art. Particularly useful leuco dyes are triarylimidazvles (as tescribed in US-A-4,089,747) and triarylmethanes (as shown in ~S-A-4,670,385). A
preferred dye-providing compo~ition iB deseribed in copending ~,S.S.N. 136,166 (filed December 18, 1987 by McClune et al), incorporated herein by reference, Once a complex has formed, it is detected to determine if the assay indicates the presence of antigen in the test 6pecimen, which would then be indicative of the presence of Streptococcal organisms. Both positive and negative control tests can be carried out simultaneously u~ing the appropriate reagents to give the desired results.
In a pre~erred embodiment of this invention, a method ~or the detection o~ Streptococcus A
comprises the steps of:
A, contacting a ~pecimen ~uspected of containing Streptococcus A with one or more extraction reagents for e~tracting antigen from the organism~
B. contacting the extracted Streptococcal A
antigen with the aqueous immunological compo~ition described herein 'ha~ing an enzyme labeled antibody, to form an e~zyme-labeled immunological complex, C. prior to, simultaneously with or subsequently to step B but after step A, contacting the extracted Streptococcal A antigen with an unlabeled antibody to the antigen, which unlabeled antibody i~ immobilized or capable of beeoming 8~, to form a comple~ of antigen and the unlabeled antibody, ~ U ~
WO90/}0232 P~TJUS90/00761 D. contacting the resulting comple~ of labeled and unlabeled antibodies with the antigen with one or more reagents which provide a dye in the pre~ence or the enzyme, and E. detecting the pre8ence of the dye as an indication of the presence of Streptococcua A in the specimen.
A~ noted in Step C of thi~ embodi~ent, the egtracted Streptococcal antigen can also be contacted with a second antiboty to the antigen, which second antibody i9 not labeled with detectable moie~ies as described above for the first antibody. This second antibody can be used to form a "sandwich" of the antigen between the two antibodies. Generally, the unlabeled antibody i6 either attached to an insoluble substrate (such as beads, membrane, filter, glass tube or other material ~nown in the art), or is capable of becoming 60 attached during the assay. In the latter instance, the unlabeled antibody could have a moiety or chemical group which readily reacts with another moiety or chemical group on the subxtrate. Thus, the second antibody could be attached to a sub trate through adsorptive, covalent or specific binding means (for e~ample, avidin-biotin).
In a pr~ferred embodiment, the unlabeled antibody iB bound in some manner to a water-insoluble microporous article ~uch as a filtratio~ membrane.
This membrane can be composed of a number of ~aterials with polyamides ~for example nylons) or modlfied polyamides being the most use~ul. It has sufficient porosity to drain uncomplexed ~aterials, but read;ly captures the immunological complex ~ormed between antigen and the two antibodies.

.~ ~ .` ?
, . ~90/10232 PCTIUS90/00761 The unlabeled antibody ~an be readily affixed to the microporous article just described.
For example, it can be affixed mechanically by çoating, ~potting or spraying and held thereon by hydrophobic bonding among molecules, or if the antibodies are attached to particles, the bonding can be between particles. It can al~o be affixed through chemical mean~. It i8 preferred that the antibodie~
be ~ound covalently to polymeric particles, which particles are mechanically a~fixed to a microporou~
filtration ~embrane of nylon.
The unlabeled antibody iE generally used in the assay in an amount sufficient to bind all available antigen to be detected.
The timing of contacting extracted antigen with the unlabeled antibody is not critical in most instances in relation to antigen contact with the labeled antibody. In a preferred embodiment where the unlabeled antibody is immobilized on a ~olid substrate, the antigen i8 contacted with it before contact with the labeled antlbody i8 aCCompliBhed.
The assay of this invention can include one or more wash ~teps in ~hich uncomple~ed or unused reagent~ are separated ~ro~ comple~ed materials.
Separation ean be accompli~hed through standard centrifugation or other mechanical means.
Preferably, uncomplexed materials are wa~hed through a ~icroporou6 ~embrane, leaving the comple~ on the ~embrane.
Other ~teps may be needed for addi~g dye-forming reagent6 or reagents for immobilizing the unlabeled antibody. One skilled in the ar~ would readily under~tand how to incorporate ~uch step~ in the assay. A representa~ive a~say iB sho~n in Example 3 below.

h~ V r~
WO90/10232 PCT/US90/00761 ,-While the assay of this invention can be carried out using 6tandard laboratory equipment and gla~sware, it i~ preferred that it be caxried out in a te~t device of some type. Various test devices are S known in the art including those described in ~S-A-3,825,410, ~S-A-3,888,629, US-A-3,970,429 and US-A-4,446,232. Particularly use~ul deYices are de~cribed in EP-A-0 308 231 and ~S-A-4,870,007.
~ ore ~pecifically, the test dev;;ce comprises a water-in~oluble 8hell having one or more test well~
therein each of which can accommodate a ~ample of a biological ~pecimen and appropriate reagents.
The shell can be prepared ~rom any useful - water-insoluble material such as glass, polymeric materials, ceramics, fibrous materials, cellulosic materials and other materials ~nown in the art.
In a preferred embodiment, the test device has three test wells designed for providing a specimen test result and positive and negative control result~. Each test well has a microporous article mounted therein. Another test device i~
described and claimed in EP-A-0 280 558. Other ~ariations of u~eful tes~ devices would be within the purview of a worker of ordinary skill in the art.
One 6uch test de~ice is currently u~ed for the SurecellTM diagnostic as~ay ~its commercially available from Ea~tman Kodak Company.
A diagno~tic kit of this invention includes the i..munological composition of this invention as well as one or more other rea~ents or rea~ent compositions. In o~e embodiment, the kit includes the im~unological composition and one or more e~traction reagents. In another embodiment, a kit includes the i~munological compo~ition and one or ~ore reagen~ for pro~iding a detectable ~pecies ~ f~' ~J i~ v woso/1o232 pcT/~sso/oo76 where the label is an enzyme. In either embodiment (where appropriate), the kit can al80 include buffer~, wash solutions, dye-providing ~olutions, test devices, extraction devices, unlabeled antibodies, immobilizing reagent8 (for e~ample avidin-coated beads), mi~ing container~, pipettes, swabs, instructions and the like.
Preferably, any test kit al80 includes a disposable test device havi~g immobilized therein an unlabeled antibody to Streptococcal antigen.
The follo~ing e2amples are provided to illustrate, but not limit, the pre~ent invention.
All percentages are by weight, unless otherwise noted.
Materials A leuco dye solution was prepared wi~h 2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-bi 6 (4-methoxy-phenyl)imidazole as follows:
Solid leuco dye (to make a 0.1% solution) was dissolved in a solution o~ 20%
poly(vinylpyrrolidone) in sodium phosphate buffer (5 mmolar). This ~olution was then added to a solution containing hydrogen peroxide ~10 mmolar~, 4'-hydro~yacetanilide (0.7 mmolar) and diethylenetriaminepenta-acetic acid (10 ~molar3 in sodium phosphate buffer to produce a final concentration of 1% poly(vinylpyrrolidone) and 0.005X leuco dye.
Succinyl~ted ca~ein wag prepared by reacting casein with an equal weight of ~uccinic anhydride for ~our hour~ at 25DC, then purifying the product by dialysis.
LoProdyneTM nylon microporous membra~e~
(Pall Corp.) were incorporated into the test wells of disposable test devices and pretreated wit~
FluoradTM FC 135 surfactant (O.OS g/m2).

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Woso/~232 PCT/US9~/00761 Anti-StreptocoCCuS A-peroxidase conjuga~e was prepared u6ing immunopurified rabbit polyclonal antibodie~ which were obtained commercially and horseradi~h perogidase by the method de~cribed by Yoshitake et al, Eu~ J. ~iochem. 101, 395 (1979).
Examples 1 & 2: IE~unolQgical ~poai~iQn~
An immunological composition for use in an assay for Streptococcal A antigen was pre!pared as follows:
A solution (1 liter) of succinylated casein (5 g) in nanopure water was prepared.
3-(N-morpholino)propanesulfonic acid buffer (20.93 g) was added to the ~olution, and its p~ was adjusted to 7.55 using sodium hydroxide.
To portions (500 ml each) of this solution were added a phenol (either 0.756 g 41-hydroxyacetanilide or 6.2 mg p-methoxyphenol), TweenTM 20 nonionic surfactant (1 g, 0.2 %) and thimerosal preservative (0.05 g). The re~ulting sQlution was filtered through a filter (0.45 ~m) and the peroxidase labeled-antibody to Streptococcus A (3.~7 ml of phosphate buffered ~aline solution containing 1.232 mg/ml antibody) was added thereto.
Thus, Example 1 contained 4'-hydroxyacetanilide as a stabilizer, and Example 2 contained p-metho~yphenol.
Example 3: AssaY for stIeptococcus ~
This e~ample demonstrates the use of the compositions of E~amples 1 and 2 in the method of 30 this invention to detect Streptococcu~ A antigen in a biological specimen.
Streptococcus A antigen waE obtained from Group A ~trep culture~ u~ing a standard nitrou~ acid e~traction procedure ~herein aqueous sodiu~ nitrite 35 was ~i~ed wikh an acidic coreagent prior to the Wos~/lo232 PCT/US9~/0076 addition of the cultured or~ani8m. The e~traction fluid was then neutralized by the addition of exce~
buffer. The Group A carbohydrate antigen was obtained using acidic ethanol and acetone, tiscarding the ~upernatant and resu~pending the pellet in 0.85%
~aline solution. The concentration of rha~nose was determined by the method of Dische and Shattle~, J.
~iQl. Chem. 1~. 595-603 (1948). This concentration (20 ~1) was resuspended ln a ~eutralized extraction ~luid comprising citric acid (10 ~1, 1.2 ~olar), sodium nitrite (120 ~1, 8 ~olar) and 4-morpholinopropane ~ulfonic acid buffer (120 ~1, 2 molar, pH 8).
A microporous membrane as described above was incorporated into each of the three test wells of ~wo disposable test devices which were aimilar to that described in EP-A-0 308 231. A dispersion of poly[styrene-co-m & ~-~2-chloroethylsulfonylmethyl)-~tyrene] beads ~2 ~1 of a 1% solid suspension in 0.1 molar glycine (pH ~.5) admixed with poly(acryl-amide) (5%) and 0.0005%, of an optica~ brightener], was added to the center area of the membrane in the test well identified as the specimen test well. To the beads were covalently bound rabbit polyclonal antibodies to Streptococcus A antigen.
A second test well of each disposable test device, considered the negative control well, contained a dried dispersion (2 ~1) of the ~ame polymeric beads to which had been attached rabbit 3~ gamma globulin ~1%, by weight o~ beads) admixed with poly(acrylamide) (5% ) and the optieal brightener.
This di~persion was applied in a center area of the ~embrane.
A third te~ well of each te~t device, - 35 considered the positive control well, contained a WO90/1~232 PCT/US~0/00761 dried disperæion of the reagent (2 ~l~ described above in poly(acrylamide) (5%), the optical brightener and 20 nglml of the Streptococcus A
antigen. Thi~ dispersion was applied in a center area of the membrane.
A sample of e~tracted antigen (200 ~l eontainin~ about 300 ng/ml of carbohydrate) was added to the specimen test well only. The peroxidase labeled antibody composition~ ~40 yl each, 9 ~g/ml conjugate) from Exampleæ 1 and 2 were added to all test wells of the disposables, followed by incubation for two minutes at room temperature. The membraneæ in the test wells were then washed twice with a wash solution (240 ~l) containing decyl ~ulfate (18 g/l) with fluid draini~g immediately.
The leuco dye eomposition (40 ~l) deæcribed above was added followed by incubation for five minutes at room temperature.
A Control composition similar to those o~
Examples 1 a~d 2, but without TweenTM 2~ nonionic surfactant, was similarly used in a Control assay for Streptococcus A. This Control compo~ition i~ like the composition described and claimed in U.S.S.N.
206,257 (noted abo~e).
The resulting æpots of dye in the center of the test wells were observed and graded on a scale of 0 ~o lO with zero representing no observable dye and lO being the highest dye den~ity. Background readings were obtained by ob~erving the preæence of any dye in the srea outside the center zone where the unlabeled antibodies were immobilized on the membranes.
The results of these teæt~ are shown in the following Table~. It i~ appare~t that the Control assay exhibited unacceptable background in the assay, ..i~W090/10232 PCT/US9~/00761 ; -especially in the negative control well. The a~say6 using the compositions of Examples 1 and 2 eghibit little or ~o background. This demon~trated ~ignificant improvement over the Control composition and as~ay.
T A B L E I (Con~rol~
T e B t W e 1 ~ 8 An~igen Ne~ative ¦ ¦ Po~itive Concen- con~Q~ en_ _ ~on~r~l.
tration Center¦ Back-¦ Center¦ Back-¦ Center¦ Back-i~ L~ rea ¦~round¦ Area ¦~round¦ ~r~ ¦gr~und 0 0 2.5 ~ 1 7 500 0 1.5 3 0.5 7 0 T A B L ~ ample 1) T e s ~ W e 1 1_~
Antigen Negative Positive Concen- Control SRecimen Control tration Center¦ Back-¦ Center¦ Back-¦ Center¦ Back-(pieo~rams) Area l~rQundl Area ¦~ound¦ Area ¦~round 0 0 1 0 0.5 7 0 500 0 0 ~ 0 7 T A B L ~ III (Ezample 2) T e 8 t W e 1 1 ~
Antigen Negative ¦ ¦ Positive Concen- Con~rol p~imen Contr~l tration Center Back- Center Back- Center Baclc-¢~ico~rams2 Area_ ~round Ar~a_ ~roun~ __e~ea ~rQund 500 0 0.5 . 4 0 7 0 The inYention has been d2scrib2d in detail ~ith par~icular reference to pre~erred embodi~ent~
thereof, but it will be understood that variations and modifications can be effec~ed within the spirit and scope of the invention.

Claims

We claim:

1. An aqueous immunological composition comprising a detectably labeled antibody to a Streptococcal antigen. and a water-soluble or water-dispersible nonionic surfactant.

2. The composition of claim 1 wherein said antibody is labeled with a radioisotope or enzyme.

3. The composition of claim 1 wherein said antibody is labeled with an enzyme.

4. The method of claim 1 wherein said antibody is directed to a Streptococcal A antigen and is labeled with peroxidase.

5. The composition of claim 1 wherein said nonionic surfactant is present in an amount of at least about 0.05 weight percent.

6. The composition of claim 1 wherein said nonionic surfactant is selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan derivatives and polyglycol ethers.

7. The composition of claim 6 wherein said nonionic surfactant is a polyoxyethylenesorbitan derivative.

8. The composition of claim 7 wherein said nonionic surfactant is polyoxyethylenesorbitan monolaurate.

9. The composition of claim 1 further comprising a buffer, a water-soluble protein or carbohydrate and a phenol.

10. An immunological diagnostic test kit useful for the determination of a Streptococcal organism comprising:
a. one or more reagents for extracting Streptococcal antigen from a Streptococcal organism, and b. an aqueous immunological composition comprising a detectably labeled antibody to composition Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant.

11. The test kit of claim 10 wherein said antibody is labeled with an enzyme, and said kit further comprises one or more reagents for providing a detectable species in the presence of said enzyme.

12. An immunological diagnostic test kit useful for the determination of a Streptococcal organism comprising:
a. an aqueous immunological composition comprising an enzyme-labeled antibody to a Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant, and b. one or more reagents for providing a detectable species in the presence of said enzyme.

13. The test kit of claim 12 further comprising a disposable test device in which is immobilized an unlabeled antibody to said Streptococcal antigen.

14. The test kit of claim 12 wherein said enzyme is peroxidase, and said one or more reagents includes a triarylimidazole leuco dye which provides a dye in the presence of peroxidase and hydrogen peroxide.

15. A method for the detection of a Streptococcal organism comprising the steps of:
A. contacting a specimen suspected of containing an extracted Streptococcal antigen with an aqueous immunological composition comprising a detectably labeled antibody to said Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant, to form a detectably labeled immunological complex, B. detecting the presence of said complex as an indication of the presence of said antigen in said specimen.

16. The method of claim 15 wherein said extracted antigen is contacted with an immobilized unlabeled antibody thereto prior to step A and after extraction.

17. The method of claim 15 wherein said antibody is labeled with an enzyme, and detection of said complex is accomplished by contacting said complex with one or more reagents which provide a detectable species in the presence of said enzyme.

18. A method for the detection of Streptococcus A comprising the steps of:
A. contacting a specimen suspected of containing Streptococcus A with one or more extraction reagents for extracting antigen from said organism, B. contacting said extracted Streptococcal A
antigen with an aqueous immunological composition comprising an enzyme-labeled antibody to said Streptococcal A antigen, and a water-soluble or water-dispersible nonionic surfactant, to form an enzyme-labeled immunological complex, C. prior to, simultaneously with or subsequently to step B but after step A, contacting said extracted Streptococcal A antigen with an unlabeled antibody to said antigen, which unlabeled antibody is immobilized or capable of becoming 80, to form a complex of antigen and said unlabeled antibody, D. contacting the resulting complex of labeled and unlabeled antibodies with said antigen with one or more reagents which provide a dye in the presence or said enzyme, and E. detecting the presence of said dye as an indication of the presence of Streptococcus A in said specimen.

19. The method of claim 18 wherein said enzyme is peroxidase, and said dye is formed using a triarylimidazole leuco dye and hydrogen peroxide.

20. The method of claim 18 wherein said nonionic surfactant is present in an amount of at least about 0.05 weight percent, and is selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan derivatives and polyglycol ethers.